In recent decades, there has been a dramatic increase in invasive mycoses, including life-threatening disseminated as well as debilitating mucosal infections. The varied condition of affected individuals, diversity of causative fungal species, and similarity between these eukaryotic pathogens and our own cells, all present significant challenges in devising effective therapies with selective toxicity. Current antifungal therapies target a narrow range of fungal components and have serious limitations including patient toxicity, efficacy, available formulations, spectrum of activity and/or the development of resistance. Our molecular studies have shown that disrupting vacuole biogenesis in the most prevalent human fungal pathogen, Candida albicans, severely impairs its capacity to colonize and invade mammalian tissue. Vacuolar function is also essential for the distantly related human pathogen, Cryptococcus neoformans, to survive within the mammalian host. In either fungus, loss of vacuolar function results in hypersensitivity to a variety of host related stresses and severely diminished expression of virulence attributes, indicating that pathogenesis is profoundly impacted on multiple levels. The absence of a closely related organelle in mammalian cells suggests that the fungal vacuole may provide an invaluable opportunity to selectively target the invading fungal pathogen, with specific chemical agents. The purpose of this study is to establish the validity of targeting the fungal vacuole as a novel strategy for antifungal therapy. Specifically, we hypothesize that the vacuole can be exploited to develop effective new antifungal therapies with low host toxicity. We have adapted a powerful high through-put assay that will enable us to efficiently screen vast chemical libraries for Vacuole Disrupting Agents (VDAs). Preliminary studies have validated this approach, and established a hit rate of ~0.44% of library compound as VDAs. In Aim 1 we will apply the screen to identify an assortment of chemically diverse compounds which disrupt the vacuole of C. albicans and/or C. neoformans. Agents active against either fungi will be considered to have 'broad spectrum' activity. VDAs will then be tested for toxicity to mammalian cells. In Aim 2, VDAs with fungal specific activity will be tested for their impact on C. albicans and C. neoformans capacity to endure host related stress, expression of virulence related attributes, and for their ability to clar either fungus from an in vitro model of host cell interaction. Finally, in Aim 3 we will use a molecular approach to define the precise functions of the C. albicans vacuole which support host colonization and pathogenesis in vivo. This will establish criteria for the selection of VDAs with the greatest antifungal efficacy.